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Secretion of the Shiga toxin B subunit (Stx1B) via an autotransporter protein optimizes the protective immune response to the antigen expressed in an attenuated E. coli (rEPEC E22ler) vaccine strain

Byrd, W;Ruiz-Perez, F;Setty, P;Zhu, C;Boedeker, EC;

We previously developed attenuated rabbit enteropathogenic E. coli (rEPEC) strains which are effective oral vaccines against their parent pathogens by deleting ler, a global regulator of virulence genes. To use these strains as orally administered vectors to deliver other antigens we incorporated the B subunit of shiga-like toxin 1(Stx1) into the passenger domain of the autotransporter EspP expressed on a plasmid. Native EspP enters the periplasm where its passenger domain is exported to the bacterial surface through an outer membrane channel formed by its translocator domain, then cleaved and secreted. Since antigen localization may determine immunogenicity, we engineered derivatives of EspP expressing Stx1B- passenger domain fusions: 1. in cytoplasm 2. in periplasm, 3. surface-attached or 4. secreted. To determine which construct was most immunogenic, rabbits were immunized with attenuated O103 E. coli strain (E22 ler) alone or expressing Stx1B in each of the above four cellular locations. IgG responses to Stx1B, and toxin-neutralizing antibodies were measured. Animals were challenged with a virulent rabbit Enterohemorrhagic E. coli (EHEC) strain of a different serogroup (O15) than the vaccine strain expressing Stx1 (RDEC-H19) and their clinical course observed. IgG responses to Stx1B subunit were induced in all animals vaccinated with the strain secreting Stx1B, in some vaccinated with surface-expressed Stx1B, but in not animals immunized with periplasmic or cytoplasmic Stx1B. Robust protection was observed only in the group immunized with the vaccine secreting Stx1B. Taken together, our data suggest that secretion of Stx1B, or other antigens, via an autotransporter, may maximize the protective response to live attenuated oral vaccine strains.